Field
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been identified as a member of the Kunitz serine proteinase inhibitor family and is hereinafter referred to as BTL.009.
Inflammatory Diseases
The inflammatory response after surgeries, trauma and infection involves neutrophil activation and infiltration into the injured tissue. The activated neutrophils release the neutral serine proteinases leukocyte elastase, cathepsin G and proteinase 3, which, if not properly controlled, cause abnormal connective tissue turnover and result in severe damage to healthy tissue (1-3, 81). The uncontrolled proteolysis can lead to a myriad of diseases including emphysema, idiopathic pulmonary fibrosis, adult respiratory distress syndrome, cystic fibrosis, rheumatoid arthritis, organ failure, and glomerulonephritis.
Proteins capable of inhibiting the neutral serine proteinases released by neutrophils can have therapeutic efficacy in treating inflammatory diseases. In patients suffering from hyperdynamic septic shock, plasma levels of the serine proteinase inhibitors antithrombin III, alpha 2-macroglobulin and inter-alpha-trypsin inhibitor, as well as those of various clotting, complement and other plasma factors, are significantly decreased (5). In an experimental endotoxemia model, the reduction in the plasma levels of these factors was considerably diminished by the intravenous injection of a soybean-derived leukocyte elastase and cathepsin G inhibitor, indicating that these neutral proteinases are at least partially responsible for the proteolysis of the plasma factors. In addition, the survival rate in the rat lethal peritonitis model (cecal ligation and puncture-induced septic shock model) was improved by treatment with the second domain of human urinary trypsin inhibitor (2), which has been shown to inhibit leukocyte elastase and cathepsin G (6, 7).
Stimulated neutrophils generate active oxygen species which contribute to inflammatory diseases, necrosis of surrounding tissues, mutagenicity and carcinogenicity (8). The most effective serine protease inhibitors in decreasing H.sub.2 O.sub.2 formation by TPA-activated neutrophils were chymotrypsin-specific inhibitors (e.g., potato inhibitor-1 and a chymotrypsin-inhibitory fragment of potato inhibitor-2), followed by bifunctional inhibitors recognizing both chymotrypsin and trypsin, and least active was soybean trypsin inhibitor, a predominantly trypsin inhibitor. In addition, cytin, a chymotrypsin- but not trypsin-specific inhibitor, significantly diminished the level of human neutrophil and monocyte activation induced by lipopolysaccharide (9).
Neutrophil chemotaxis also plays an important role in the inflammatory response and, when excessive or persistent, may augment tissue damage (10). Inhibitors of cathepsin G and chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose.
IL-1, a proinflammatory cytokine, is secreted from monocytes at inflammatory sites as an inactive precursor. Leukocyte elastase and cathepsin G cleave the IL-1 precursor to form fully active forms of IL-1 (11). Synovial fluid collected from patients with inflammatory polyarthritis and bronchoalveolar lavage fluid from patients with sarcoidosis process the IL-1 precursor into the same active forms as leukocyte elastase and cathepsin G. Control fluids from patients who had no symptoms of inflammatory disease did not exhibit the processing activity. Only lavage fluids that processed precursor IL-1 contain cathepsin G and/or elastase activity.
Synthetic tannin exhibits anti-inflammatory properties in skin diseases. Tannin specifically inhibits leukocyte elastase in an irreversible manner and it is believed that the anti-inflammatory properties of synthetic tannin may at least in part be due to inactivation of elastase (12).
Lung Injury
Many syndromes of lung injury, including emphysema, adult respiratory distress syndrome, cystic fibrosis and idiopathic pulmonary fibrosis, are associated with accumulation of neutrophils within the pulmonary parenchyma. Activated neutrophils have the capacity to produce lung injury by secreting products including proteinases and reactive oxygen species (13). Neutral serine proteinases secreted from activated neutrophils are capable of inducing damage to lung alveolar extracellular matrix (ECM) by directly digesting the matrix and through the activation of latent metalloproteases resident in the matrix (14). Proteinase 3 and leukocyte elastase have been shown to cause significant lung damage and emphysema when administered by tracheal insuffilation or injection to hamsters (16, 17).
Inhibitors of neutrophil neutral serine proteinases have been shown to exert potent therapeutic effects on pulmonary emphysema, adult respiratory distress syndrome and other diseases involving tissue degradation. Treatment of hamsters with Eglin c, a neutral serine proteinase inhibitor, completely protected hamsters against leukocyte elastase-induced emphysema (18). Derivatives of 5-methyl-4H-3,1-benzoxazin-4-one, shown to be highly specific inhibitors of leukocyte elastase, efficiently prevented degradation of insoluble elastin by stimulated neutrophils (19). These small molecule inhibitors also significantly suppressed leukocyte induced pulmonary hemorrhage and emphysema in hamsters (19). Alpha 1-proteinase inhibitor and soybean trypsin inhibitor, two leukocyte elastase and cathepsin G inhibitors, were also shown to completely or nearly completely inhibit neutrophil-induced ECM solubilization (13).
However, alpha 1-proteinase inhibitor, the major endogenous serine proteinase inhibitor for neutrophil elastase, is easily inactivated by proteolysis by metalloproteinases present in the injured lung and by oxidation (20, 21). Oxidative inactivation of alpha 1-proteinase has been linked to the pathogenesis of pulmonary emphysema associated with cigarette smoking (22).
Vascular Effects
Injury to the vascular endothelium, such as that occurs during angioplasty, can result in the accumulation of neutrophils and platelets and platelet activation at the site of injury. Platelet accumulation and activation at the injured site can result in abrupt artery closure. Cathepsin G potently induces platelet aggregation, secretion and calcium mobilization by binding to a specific receptor on platelets (23). Leukocyte elastase, though having no platelet agonist activity itself, increases the apparent affinity of cathepsin G binding to platelets and enhances cathepsin G-induced platelet activation. Thrombospondin 1, which inactivates cathepsin G by binding near the enzyme's active site, protected fibronectin from cleavage by cathepsin G and blocked cathepsin G-mediated platelet aggregation (24).
Endothelin-1 (ET-1) is potent vasoconstrictor peptide secreted by endothelial cells. Marked ET-1 degradation is observed in the presence of activated neutrophils. ET-1 inactivation could play a role in acute inflammatory reactions where neutrophils adhere to the vascular endothelial cells. Soybean trypsin inhibitor abolishes ET-1 degradation almost completely, suggesting a role of cathepsin G in ET-1 hydrolysis (25). Among the purified leukocyte enzymes tested, cathepsin G hydrolyzed ET-1 at the highest rate.
Cathepsin G converts angiotensinogen and angiotensin I to angiotensin II (26, 27). The neutrophil-angiotensin system does not require renin or converting enzyme and may function as a mobile effector pathway which modulates tissue blood flow and/or vascular permeability.
Proteinase Inhibitor Structure and Specificity
Cathepsin G, leukocyte elastase and proteinase 3 are neutral serine proteinases that exist primarily in azurophilic granules of neutrophils. Elastase has a preference for hydrophobic (e.g., neutral) residues at the P1 site such as valine, alanine, isoleucine and leucine (28, 29). (The reactive-site sequence of proteinase inhibitors and substrates are written as . . . -P3-P2-P1-P'1-P'2-P'3- . . . , where-P1-P'1-denotes the reactive site). Cathepsin G has a similar preference for large hydrophobic residues (i.e., phenylalanine, leucine) and basic residues (lysine, arginine) and exhibits dual and equal trypsin- and chymotrypsin-like specificities (30). Proteinase 3 prefers small aliphatic amino acids such as alanine, serine and valine at the P1 site (15, 31). The P3-S3 interaction during human leukocyte elastase hydrolysis of peptide substrates has also been determined to be important (32). (S3 refers to the residue on the inhibited proteinase that interacts with the P3 residue on the inhibitor.)
Kunitz Inhibitors
Protein inhibitors of serine proteinases can be grouped into several families, including the Kunitz, serpin, Kazal, and mucous protein inhibitor families, based on conserved structural features. Members of each family exhibit greatly varied binding specificities, and members of different families can have similar inhibitory profiles. The binding specificities of the proteinase inhibitors are determined by the residue at the P1 position as well as other residues that lie at the interface between the inhibitor and the bound target proteinase. The P1 residue in Kunitz domain proteins lies immediately C-terminal to the conserved second cysteine (position 15; aprotinin numbering).
All members of the Kunitz domain protein family have the same number (six) and spacing of cysteine residues. The precise bonding of cysteine residues to form the three intrachain disulfide bonds is known and invariant for all previously known Kunitz members (33).
Members of the Kunitz domain protein family function as inhibitors of serine proteases. Each inhibitor has a unique inhibition specificity profile towards the serine proteases. However, inhibitors with a basic residue (i.e., arginine or lysine) immediately following the second cysteine residue tend to have greater potencies towards proteases that cleave proteins at basic residues. In addition, mutation of the lysine residue at this position in aprotinin to a valine resulted in a dramatic increase in the protein's potency towards neutrophil elastase, a protease that typically cleaves proteins at residues with small neutral aliphatic side chains (34).
The serine protease inhibitory activities of the Kunitz domain proteins has led to their evaluation as potential therapeutics in a number of disease indications. For example, aprotinin is a potent inhibitor of proteases involved in the blood clotting cascade and is used clinically to reduce bleeding during open heart surgery (35). Human placental bikunin is a potent inhibitor of plasmin, which has been implicated in facilitating metastasis and tumor growth (36). Other disease indications in which serine proteases are believed to play a significant pathological role and the Kunitz domain proteins may therefore be effective therapeutics include traumatic brain injury and stroke (37, 38), cystic fibrosis (39, 40), emphysema (41), arthritis and anemia (42) and non-insulin dependent diabetes (43).
Kunitz domains that exist within larger proteins have been shown to retain their functional activities when produced as single domains (44). Kunitz-type inhibitors have been described in the patent literature (85).
Serine proteinase inhibitors of the Kunitz family typically exhibit significantly tighter binding to trypsin and chymotrypsin, two proteases with relatively strict P1 specificities (trypsin=arginine, lysine; chymotrypsin=tyrosine, phenylalanine, tryptophan) but with few restrictions at other P and P' positions, than to the three neutral proteinases secreted by neutrophils. For example, aprotinin is a potent inhibitor of trypsin (Ki=0.02 nM) and chymotrypsin (Ki=1.3 nM) but does not inhibit leukocyte elastase (44). Similarly, placental bikunin inhibits trypsin (Ki=0.01 nM) and chymotrypsin (Ki=0.48 nM) but not leukocyte elastase (44). Tissue factor pathway inhibitor (TFPI), another member of the Kunitz family, inhibits trypsin (0.1 nM) and chymotrypsin (Ki=0.75 nM) but is a weak inhibitor of leukocyte elastase (Ki=400 nM) and cathepsin G (Ki=100-200 nM) (45, 46). In addition, these Kunitz family members exhibit potent inhibitory activity towards serine proteinases having trypsin-like substrate specificity involved in both coagulation and fibrinolysis (44-47). Elastase and cathepsin G have been reported to proteolytically cleave and inactivate TFPI (4, 46).
Human inter-alpha-trypsin inhibitor (I alpha I), a plasma Kunitz family proteinase inhibitor, is a potent inhibitor of trypsin (Ki=0.078 nM) and chymotrypsin (1. 1 nM) but exhibits somewhat lesser activity against cathepsin G (Ki=18 nM) and leukocyte elastase (Ki=61 nM) (6). Similarly, a Kunitz-type inhibitor purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes potently inhibited trypsin (Ki=0.46 nM) and chymotrypsin (Ki=5.5 nM), but was somewhat less active towards leukocyte elastase (Ki=72 nM) (48).
Soybean trypsin inhibitor (STI) is a potent Kunitz family inhibitor of tryspin but a significantly weaker inhibitor of chymotrypsin (Ki(1)=1000 nM; Ki(2)=300 nM) (49). STI has been reported to exhibit similar inhibitory activity towards chymotrypsin and leukocyte elastase (25). On the other hand, a serine protease inhibitor from larvae of parasitic nematode Anisakis simplex that has 96% amino acid identity to soybean trypsin inhibitor was reported to inhibit trypsin and elastase but not chymotrypsin (50). In addition, a Kunitz-type inhibitor purified from potato tubers (Solanum tuberosum L) was reported to be an effective inhibitor of trypsin, leukocyte elastase, and chymotrypsin (51).
Non-Kunitz Proteinase Inhibitors
Numerous serine proteinase inhibitors from families other than that of the Kunitz family have been reported to inhibit neutral serine proteinases, including those secreted by activated neutrophils. Alpha-1-proteinase and alpha-2-macroglobulin, members of the serpin proteinase inhibitor family, inhibit elastase, cathepsin G and proteinase 3 (15, 52-55). Alpha-1-proteinase has been described as the major serum inhibitor of elastase and cathepsin G (54). Alpha-1-antichymotrypsin, another serpin family proteinase inhibitor, inhibits cathepsin G (53, 56, 55) but not proteinase 3 (15), and has been described as another physiological cathepsin G inhibitor (53). Monocyte/neutrophil elastase inhibitor, also a serpin family inhibitor, inhibits elastase and proteinase 3 (57). Antileukoproteinase (SLPI) and elafin, members of the mucous proteinase inhibitor family, inhibit elastase (Ki=0.6 mM) (58, 59) but not proteinase 3 (15) and cathepsin G (58). Eglin C, a member of the potato inhibitor 1 family from leech Hirudo medicinalis, inhibits leukocyte elastase (Ki=0.37 nM) and cathepsin G (Ki.about.0.1 nM) (60-62) but only weakly inhibits proteinase 3 (15).